Program in Macromolecular Structure, Motion, Control

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RNA splicing is the removal of an intron and the simultaneous ligation of its flanking exons in the generation of mature cellular RNA molecules. Splicing provides a critical level of genetic control. Through alternative splicing, the proteome of a higher eukaryote is substantially more complex than the number of genes in its genome. The importance of RNA splicing to human health is manifest by the observation that at least 15% of point mutations leading to heritable human diseases cause defects in pre-mRNA splicing. While most introns are removed by the spliceosome, some introns are able to catalyze their own removal from the primary transcript. The discovery of the group I class of introns provided the first indication that not all enzymes are proteins. Their existence proves that RNA can select 5' and 3' splice sites and catalyze the two transesterification reactions of intron removal. Our understanding of the structural basis of RNA splicing is still in its infancy, but in 2004, almost 25 years after its initial discovery, the first crystal structure of a complete group I intron in complex with both its 5' and 3' exons was finally determined. This result will now be exploited to achieve several important objectives relevant to the structural basis of RNA splicing and the dynamic conformational rearrangements that occur during the splicing process. The overriding goals of these studies are: (i) to determine the X-ray crystal structure of each step in an RNA splicing pathway, (ii) to explain how RNA tertiary structure is formed and active sites created in the absence of proteins, (iii) to reveal how metal ions contribute to RNA catalysis and how alteration of ligands affects metal ion specificity, (iv) to visualize the nature of the transition state of the phosphoryl transfer reaction promoted during the 5'-exon cleavage and exon ligation reactions, and (v) to explore how group I intron splicing is facilitated by protein cofactors. Many ribonucleoprotein complexes are expected to undergo complex conformational changes during their function, so understanding how an RNA is reconfigured during a multi-step splicing reaction will provide a valuable precedent for considering these complex dynamic processes. RNA enzymes, or ribozymes, are the molecules most likely to be the progenitors of modern biological catalysts, and understanding how they promote their reactions will provide critical insight into enzymological function. This series of structural snapshots will reveal principles of RNA folding, structural dynamics, and metal mediated catalysis, principles that are certain to have parallels in most cellular machines that include RNA.